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Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily

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ABSTRACT

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

No MeSH data available.


Graphical representation of combined ChIP-Seq, ChIP-exo and dRNA-seq for four class 1 targets.Each target has the relative position of ChIP-exo (blue line) peak centre (dotted line) and putative transcriptional start site (TSS - solid arrow) indicated with the distance in bp (black numbers) relative to the down stream start codon of target genes. The y-axis scale corresponds to number of reads for ChIP data with each window corresponding to 200 bp with each ChIP-peak being ~50 bp wide. Above each is the relative binding site sequence coloured following the weblogo scheme (A – red, T – green, C – blue and G – yellow) from the MEME results.
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f6: Graphical representation of combined ChIP-Seq, ChIP-exo and dRNA-seq for four class 1 targets.Each target has the relative position of ChIP-exo (blue line) peak centre (dotted line) and putative transcriptional start site (TSS - solid arrow) indicated with the distance in bp (black numbers) relative to the down stream start codon of target genes. The y-axis scale corresponds to number of reads for ChIP data with each window corresponding to 200 bp with each ChIP-peak being ~50 bp wide. Above each is the relative binding site sequence coloured following the weblogo scheme (A – red, T – green, C – blue and G – yellow) from the MEME results.

Mentions: MEME analysis of the ChIP-seq data detected only 14 class 1 (11-3-11 bp inverted repeat) sites out of the 117 target sites bound by RsrR on the S. venezuelae chromosome. However, ChIP-Seq and EMSAs show that RsrR can bind to target genes whether they contain class 1 or class 2 sites. This differs from E. coli NsrR which binds only weakly to target sites containing putative half sites (class 2)28. To gain more information about RsrR recognition sequences and the positions of these binding sites at target promoters we combined differential RNA-seq (dRNA-seq, accession number GSE81104), which maps the start sites of all expressed transcripts, with ChIP-exo (accession number GSE80818) which uses Lambda exonuclease to trim excess DNA away from ChIP complexes leaving only the DNA which is actually bound and protected by RsrR. For dRNA-seq, total RNA was prepared from cultures of wild type S. venezuelae and for the ∆rsrR mutant grown for 16 hours. ChIP-exo was performed on the ∆rsrR strain producing Flag-tagged RsrR, also at 16 hours. ChIP-exo identified 630 binding sites which included the 117 targets identified previously using ChIP-seq. The ChIP-exo peaks are on average only ~50 bp wide giving much better resolution of the RsrR binding sites at each target. MEME analysis using all 630 ChIP-exo sequences identified the class 2 binding motif in every sequence and we identified transcript start sites (TSS) for 261 of the 630 RsrR target genes using our dRNA-seq data (Supplementary data S1). Figure 6 shows a graphical representation of class 1 targets that have clearly defined TSS, indicating the centre of the ChIP peak, the associated TSS and any genes within the ~200 bp frame. Based on the RsrR binding site position at putative target genes RsrR likely acts as both a transcriptional activator and repressor and we have shown that RsrR represses transcription of sven6562 which is a class 1 target with two 11-3-11 bp binding site in the intergenic region between sven6562 and rsrR. The functional significance of RsrR binding to the other class 1 and 2 target genes identified here by ChIP-seq and ChIP-exo remains to be seen but they are not significantly affected by loss of RsrR under the conditions used in our experiments.


Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily
Graphical representation of combined ChIP-Seq, ChIP-exo and dRNA-seq for four class 1 targets.Each target has the relative position of ChIP-exo (blue line) peak centre (dotted line) and putative transcriptional start site (TSS - solid arrow) indicated with the distance in bp (black numbers) relative to the down stream start codon of target genes. The y-axis scale corresponds to number of reads for ChIP data with each window corresponding to 200 bp with each ChIP-peak being ~50 bp wide. Above each is the relative binding site sequence coloured following the weblogo scheme (A – red, T – green, C – blue and G – yellow) from the MEME results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5015018&req=5

f6: Graphical representation of combined ChIP-Seq, ChIP-exo and dRNA-seq for four class 1 targets.Each target has the relative position of ChIP-exo (blue line) peak centre (dotted line) and putative transcriptional start site (TSS - solid arrow) indicated with the distance in bp (black numbers) relative to the down stream start codon of target genes. The y-axis scale corresponds to number of reads for ChIP data with each window corresponding to 200 bp with each ChIP-peak being ~50 bp wide. Above each is the relative binding site sequence coloured following the weblogo scheme (A – red, T – green, C – blue and G – yellow) from the MEME results.
Mentions: MEME analysis of the ChIP-seq data detected only 14 class 1 (11-3-11 bp inverted repeat) sites out of the 117 target sites bound by RsrR on the S. venezuelae chromosome. However, ChIP-Seq and EMSAs show that RsrR can bind to target genes whether they contain class 1 or class 2 sites. This differs from E. coli NsrR which binds only weakly to target sites containing putative half sites (class 2)28. To gain more information about RsrR recognition sequences and the positions of these binding sites at target promoters we combined differential RNA-seq (dRNA-seq, accession number GSE81104), which maps the start sites of all expressed transcripts, with ChIP-exo (accession number GSE80818) which uses Lambda exonuclease to trim excess DNA away from ChIP complexes leaving only the DNA which is actually bound and protected by RsrR. For dRNA-seq, total RNA was prepared from cultures of wild type S. venezuelae and for the ∆rsrR mutant grown for 16 hours. ChIP-exo was performed on the ∆rsrR strain producing Flag-tagged RsrR, also at 16 hours. ChIP-exo identified 630 binding sites which included the 117 targets identified previously using ChIP-seq. The ChIP-exo peaks are on average only ~50 bp wide giving much better resolution of the RsrR binding sites at each target. MEME analysis using all 630 ChIP-exo sequences identified the class 2 binding motif in every sequence and we identified transcript start sites (TSS) for 261 of the 630 RsrR target genes using our dRNA-seq data (Supplementary data S1). Figure 6 shows a graphical representation of class 1 targets that have clearly defined TSS, indicating the centre of the ChIP peak, the associated TSS and any genes within the ~200 bp frame. Based on the RsrR binding site position at putative target genes RsrR likely acts as both a transcriptional activator and repressor and we have shown that RsrR represses transcription of sven6562 which is a class 1 target with two 11-3-11 bp binding site in the intergenic region between sven6562 and rsrR. The functional significance of RsrR binding to the other class 1 and 2 target genes identified here by ChIP-seq and ChIP-exo remains to be seen but they are not significantly affected by loss of RsrR under the conditions used in our experiments.

View Article: PubMed Central - PubMed

ABSTRACT

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

No MeSH data available.